Cryo-EM structure of the CMG Fork Protection Complex at a replication fork

2024-05-22 REL PDBe PDBe PDBe 2019-08-16 2020-05-06 2020-05-06 2024-05-22 Medical Research Council (MRC, United Kingdom) MC_UP_1201/12 United Kingdom Cryo-EM structure of the CMG Fork Protection Complex at a replication fork - Conformation 1 Baretic D Jenkyn-Bedford M protein-DNA complex, replisome, AAA+ helicase, CMG, GINS, fork DNA, MCM, fork protection complex, CIP-box, REPLICATION Baretic D Jenkyn-Bedford M Aria V Cannone G Skehel M Yeeles JTP Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork. Mol.Cell US 78 926 940.e13 2020 32369734 doi:10.1016/j.molcel.2020.04.012 1097-2765 2168 MOCEFL 6skl FULLOVERLAP Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1.4 CMG-Csm3-Tof1-Mrc1-Ctf4 1 1 2 3 4 5 6 7 8 9 10 11 12 15 16 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) DNA 1 13 14 synthetic construct DNA replication licensing factor MCM2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.098911539 1 Saccharomyces cerevisiae LEVO MSDNRRRRREEDDSDSENELPPSSPQQHFRGGMNPVSSPIGSPDMINPEGDDNEVDDVPDIDEVEEQMNEVDLMDDNMYE DYAADHNRDRYDPDQVDDREQQELSLSERRRIDAQLNERDRLLRNVAYIDDEDEEQEGAAQLDEMGLPVQRRRRRRQYED LENSDDDLLSDMDIDPLREELTLESLSNVKANSYSEWITQPNVSRTIARELKSFLLEYTDETGRSVYGARIRTLGEMNSE SLEVNYRHLAESKAILALFLAKCPEEMLKIFDLVAMEATELHYPDYARIHSEIHVRISDFPTIYSLRELRESNLSSLVRV TGVVTRRTGVFPQLKYVKFNCLKCGSILGPFFQDSNEEIRISFCTNCKSKGPFRVNGEKTVYRNYQRVTLQEAPGTVPPG RLPRHREVILLADLVDVSKPGEEVEVTGIYKNNYDGNLNAKNGFPVFATIIEANSIKRREGNTANEGEEGLDVFSWTEEE EREFRKISRDRGIIDKIISSMAPSIYGHRDIKTAVACSLFGGVPKNVNGKHSIRGDINVLLLGDPGTAKSQILKYVEKTA HRAVFATGQGASAVGLTASVRKDPITKEWTLEGGALVLADKGVCLIDEFDKMNDQDRTSIHEAMEQQSISISKAGIVTTL QARCSIIAAANPNGGRYNSTLPLAQNVSLTEPILSRFDILCVVRDLVDEEADERLATFVVDSHVRSHPENDEDREGEELK NNGESAIEQGEDEINEQLNARQRRLQRQRKKEEEISPIPQELLMKYIHYARTKIYPKLHQMDMDKVSRVYADLRRESIST GSFPITVRHLESILRIAESFAKMRLSEFVSSYDLDRAIKVVVDSFVDAQKVSVRRQLRRSFAIYTLGH P29469 3.6.4.12 DNA replication licensing factor MCM3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.107653508 1 Saccharomyces cerevisiae LEVO MEGSTGFDGDATTFFAPDAVFGDRVRRFQEFLDTFTSYRDSVRSIQVYNSNNAANYNDDQDDADERDLLGDDDGDDLEKE KKAASSTSLNILPHRIIISLDDLREFDRSFWSGILVEPAYFIPPAEKALTDLADSMDDVPHPNASAVSSRHPWKLSFKGS FGAHALSPRTLTAQHLNKLVSVEGIVTKTSLVRPKLIRSVHYAAKTGRFHYRDYTDATTTLTTRIPTPAIYPTEDTEGNK LTTEYGYSTFIDHQRITVQEMPEMAPAGQLPRSIDVILDDDLVDKTKPGDRVNVVGVFKSLGAGGMNQSNSNTLIGFKTL ILGNTVYPLHARSTGVAARQMLTDFDIRNINKLSKKKDIFDILSQSLAPSIYGHDHIKKAILLMLMGGVEKNLENGSHLR GDINILMVGDPSTAKSQLLRFVLNTASLAIATTGRGSSGVGLTAAVTTDRETGERRLEAGAMVLADRGVVCIDEFDKMTD VDRVAIHEVMEQQTVTIAKAGIHTTLNARCSVIAAANPVFGQYDVNRDPHQNIALPDSLLSRFDLLFVVTDDINEIRDRS ISEHVLRTHRYLPPGYLEGEPVRERLNLSLAVGEDADINPEEHSNSGAGVENEGEDDEDHVFEKFNPLLQAGAKLAKNKG NYNGTEIPKLVTIPFLRKYVQYAKERVIPQLTQEAINVIVKNYTDLRNDDNTKKSPITARTLETLIRLATAHAKVRLSKT VNKVDAKVAANLLRFALLGEDIGNDIDEEESEYEEALSKRSPQKSPKKRQRVRQPASNSGSPIKSTPRRSTASSVNATPS SARRILRFQDDEQNAGEDDNDIMSPLPADEEAELQRRLQLGLRVSPRRREHLHAPEEGSSGPLTEVGTPRLPNVSSAGQD DEQQQSVISFDNVEPGTISTGRLSLISGIIARLMQTEIFEEESYPVASLFERINEELPEEEKFSAQEYLAGLKIMSDRNN LMVADDKVWRV P24279 3.6.4.12 DNA replication licensing factor MCM4 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.10513837499999999 1 Saccharomyces cerevisiae LEVO MSQQSSSPTKEDNNSSSPVVPNPDSVPPQLSSPALFYSSSSSQGDIYGRNNSQNLSQGEGNIRAAIGSSPLNFPSSSQRQ NSDVFQSQGRQGRIRSSASASGRSRYHSDLRSDRALPTSSSSLGRNGQNRVHMRRNDIHTSDLSSPRRIVDFDTRSGVNT LDTSSSSAPPSEASEPLRIIWGTNVSIQECTTNFRNFLMSFKYKFRKILDEREEFINNTTDEELYYIKQLNEMRELGTSN LNLDARNLLAYKQTEDLYHQLLNYPQEVISIMDQTIKDCMVSLIVDNNLDYDLDEIETKFYKVRPYNVGSCKGMRELNPN DIDKLINLKGLVLRSTPVIPDMKVAFFKCNVCDHTMAVEIDRGVIQEPARCERIDCNEPNSMSLIHNRCSFADKQVIKLQ ETPDFVPDGQTPHSISLCVYDELVDSCRAGDRIEVTGTFRSIPIRANSRQRVLKSLYKTYVDVVHVKKVSDKRLDVDTST IEQELMQNKVDHNEVEEVRQITDQDLAKIREVAAREDLYSLLARSIAPSIYELEDVKKGILLQLFGGTNKTFTKGGRYRG DINILLCGDPSTSKSQILQYVHKITPRGVYTSGKGSSAVGLTAYITRDVDTKQLVLESGALVLSDGGVCCIDEFDKMSDS TRSVLHEVMEQQTISIAKAGIITTLNARSSILASANPIGSRYNPNLPVTENIDLPPPLLSRFDLVYLVLDKVDEKNDREL AKHLTNLYLEDKPEHISQDDVLPVEFLTMYISYAKEHIHPIITEAAKTELVRAYVGMRKMGDDSRSDEKRITATTRQLES MIRLAEAHAKMKLKNVVELEDVQEAVRLIRSAIKDYATDPKTGKIDMNLVQTGKSVIQRKLQEDLSREIMNVLKDQASDS MSFNELIKQINEHSQDRVESSDIQEALSRLQQEDKVIVLGEGVRRSVRLNNRV P30665 3.6.4.12 Minichromosome maintenance protein 5 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.08650573399999999 1 Saccharomyces cerevisiae LEVO MSFDRPEIYSAPVLQGESPNDDDNTEIIKSFKNFILEFRLDSQFIYRDQLRNNILVKNYSLTVNMEHLIGYNEDIYKKLS DEPSDIIPLFETAITQVAKRISILSRAQSANNNDKDPENTSMDTDSLLLNSLPTFQLILNSNANQIPLRDLDSEHVSKIV RLSGIIISTSVLSSRATYLSIMCRNCRHTTSITINNFNSITGNTVSLPRSCLSTIESESSMANESNIGDESTKKNCGPDP YIIIHESSKFIDQQFLKLQEIPELVPVGEMPRNLTMTCDRYLTNKVIPGTRVTIVGIYSIYNSKNGAGSGRSGGGNGGSG VAIRTPYIKILGIQSDVETSSIWNSVTMFTEEEEEEFLQLSRNPKLYEILTNSIAPSIFGNEDIKKAIVCLLMGGSKKIL PDGMRLRGDINVLLLGDPGTAKSQLLKFVEKVSPIAVYTSGKGSSAAGLTASVQRDPMTREFYLEGGAMVLADGGVVCID EFDKMRDEDRVAIHEAMEQQTISIAKAGITTVLNSRTSVLAAANPIYGRYDDLKSPGDNIDFQTTILSRFDMIFIVKDDH NEERDISIANHVINIHTGNANAMQNQQEENGSEISIEKMKRYITYCRLKCAPRLSPQAAEKLSSNFVTIRKQLLINELES TERSSIPITIRQLEAIIRITESLAKLELSPIAQERHVDEAIRLFQASTMDAASQDPIGGLNQASGTSLSEIRRFEQELKR RLPIGWSTSYQTLRREFVDTHRFSQLALDKALYALEKHETIQLRHQGQNIYRSGV P29496 3.6.4.12 DNA replication licensing factor MCM6 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.11311021099999999 1 Saccharomyces cerevisiae LEVO MSSPFPADTPSSNRPSNSSPPPSSIGAGFGSSSGLDSQIGSRLHFPSSSQPHVSNSQTGPFVNDSTQFSSQRLQTDGSAT NDMEGNEPARSFKSRALNHVKKVDDVTGEKVREAFEQFLEDFSVQSTDTGEVEKVYRAQIEFMKIYDLNTIYIDYQHLSM RENGALAMAISEQYYRFLPFLQKGLRRVVRKYAPELLNTSDSLKRSEGDEGQADEDEQQDDDMNGSSLPRDSGSSAAPGN GTSAMATRSITTSTSPEQTERVFQISFFNLPTVHRIRDIRSEKIGSLLSISGTVTRTSEVRPELYKASFTCDMCRAIVDN VEQSFKYTEPTFCPNPSCENRAFWTLNVTRSRFLDWQKVRIQENANEIPTGSMPRTLDVILRGDSVERAKPGDRCKFTGV EIVVPDVTQLGLPGVKPSSTLDTRGISKTTEGLNSGVTGLRSLGVRDLTYKISFLACHVISIGSNIGASSPDANSNNRET ELQMAANLQANNVYQDNERDQEVFLNSLSSDEINELKEMVKDEHIYDKLVRSIAPAVFGHEAVKKGILLQMLGGVHKSTV EGIKLRGDINICVVGDPSTSKSQFLKYVVGFAPRSVYTSGKASSAAGLTAAVVRDEEGGDYTIEAGALMLADNGICCIDE FDKMDISDQVAIHEAMEQQTISIAKAGIHATLNARTSILAAANPVGGRYNRKLSLRGNLNMTAPIMSRFDLFFVILDDCN EKIDTELASHIVDLHMKRDEAIEPPFSAEQLRRYIKYARTFKPILTKEARSYLVEKYKELRKDDAQGFSRSSYRITVRQL ESMIRLSEAIARANCVDEITPSFIAEAYDLLRQSIIRVDVDDVEMDEEFDNIESQSHAASGNNDDNDDGTGSGVITSEPP ADIEEGQSEATARPGTSEKKKTTVTYDKYVSMMNMIVRKIAEVDREGAEELTAVDIVDWYLLQKENDLGSLAEYWEERRL AFKVIKRLVKDRILMEIHGTRHNLRDLENEENENNKTVYVIHPNCEVLDQLEPQDSS P53091 3.6.4.12 DNA replication licensing factor MCM7 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.09504987499999999 1 Saccharomyces cerevisiae LEVO MSAALPSIQLPVDYNNLFNEITDFLVTFKQDTLSSDATRNENEDENLDAENIEQHLLEKGPKYMAMLQKVANRELNSVII DLDDILQYQNEKFLQGTQADDLVSAIQQNANHFTELFCRAIDNNMPLPTKEIDYKDDVLDVILNQRRLRNERMLSDRTNE IRSENLMDTTMDPPSSMNDALREVVEDETELFPPNLTRRYFLYFKPLSQNCARRYRKKAISSKPLSVRQIKGDFLGQLIT VRGIITRVSDVKPAVEVIAYTCDQCGYEVFQEVNSRTFTPLSECTSEECSQNQTKGQLFMSTRASKFSAFQECKIQELSQ QVPVGHIPRSLNIHVNGTLVRSLSPGDIVDVTGIFLPAPYTGFKALKAGLLTETYLEAQFVRQHKKKFASFSLTSDVEER VMELITSGDVYNRLAKSIAPEIYGNLDVKKALLLLLVGGVDKRVGDGMKIRGDINVCLMGDPGVAKSQLLKAICKISPRG VYTTGKGSSGVGLTAAVMKDPVTDEMILEGGALVLADNGICCIDEFDKMDESDRTAIHEVMEQQTISISKAGINTTLNAR TSILAAANPLYGRYNPRLSPLDNINLPAALLSRFDILFLMLDIPSRDDDEKLAEHVTYVHMHNKQPDLDFTPVEPSKMRE YIAYAKTKRPVMSEAVNDYVVQAYIRLRQDSKREMDSKFSFGQATPRTLLGIIRLSQALAKLRLADMVDIDDVEEALRLV RVSKESLYQETNKSKEDESPTTKIFTIIKKMLQETGKNTLSYENIVKTVRLRGFTMLQLSNCIQEYSYLNVWHLINEGNT LKFVDDGTMDTDQEDSLVSTPKLAPQTTASANVSAQDSDIDLQDA P38132 3.6.4.12 DNA replication complex GINS protein PSF1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.024230576 1 Saccharomyces cerevisiae LEVO MYGDLGNKLVLEAKRTKQLYARSNQDVNLPMYHEDIIRNILKEVSNLRKNTEYLKEQQQLGMLDDKVAKCQYFVTLLCME RNKRCLLAYQRLRTDILDSMAWNNNGLDLMSSITFSQQDTNNLSHQEQEYLKEYCDLITDLKSGDLVDIDLSGSLVPPSD VFIDVRVLKDAGEIQTEYGVFNLIKDSQFFVRQSDVERLIQQGYLQKI Q12488 DNA replication complex GINS protein PSF2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.025096807 1 Saccharomyces cerevisiae LEVO MSLPAHLQQTFSPEEIQFIVENEPIKIFPRITTRQKIRGDDRGTGNHTRWQLITTDDKALNNMVAMRSTEVVLWIALLLK QQSKCSIVAPQWLTTKELDRKIQYEKTHPDRFSELPWNWLVLARILFNKAKDDFHDPIHELRGKIQDLREIRQIKVLKGL KYLNESHLQLDNLSLLEINELRPFITEIMDKLREIHTASLTAGTENDEEEFNI P40359 DNA replication complex GINS protein PSF3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.024437859

His-tag at the N-terminus (24 residues)

1 Saccharomyces cerevisiae LEVO MGSSHHHHHHSSGLVPRGSHMASMGYYDIDDVLADGTEFPCKFQYDIPGLGYLENNPGRPITKNTKLSLPLWLARILAIV GGDEALVDEEPVPFVELLPPDMFSTKVMNAIKTDPVALDLHSINSHFFSLAIKWIMLFSEKELANVVSELLLQRAQELNH HASSLSIDLNADSTGKNSANTNIATSTFLLKLEEMEKEIYKKSHESYKDTKRWMFKK Q12146 DNA replication complex GINS protein SLD5 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.033983616999999994 1 Saccharomyces cerevisiae LEVO MDINIDDILAELDKETTAVDSTKITQGSSSTTHRDANTIVGSSLDLNDKTQIYVSPQQDFSDLMKSWKNERCSPELLPYP HQLMKRLLNRISMQSQLIENISMGFLDMQNASNANPPMPNESKLPLLCMETELERLKFVIRSYIRCRLSKIDKFSLYLRQ LNEDENSLISLTDLLSKDEIKYHDTHSLIWLKLVNDSILKYMPEELQAINDTEGSVNMIDEPDWNKFVFIHVNGPPDGKW NEDPLLQENEFGKPCYTVTIPDLKEEVELTIGSIYVMRYEVIRDLLRDDKVALI Q03406 Cell division control protein 45 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.07432483599999999 1 Saccharomyces cerevisiae LEVO MYYGISQFSEAYNKILRNSSSHSSCQLVIFVSCLNIDALCATKMLSLLFKKQLVQSQIVPIFGYSELRRHYSQLDDNINS LLLVGFGGVIDLEAFLEIDPQEYVIDTDEKSGEQSFRRDIYVLDAHRPWNLDNIFGSQIIQCFDDGTVDDTLGEQKEAYY KLLELDEESGDDELSGDENDNNGGDDEATDADEVTDEDEEDEDETISNKRGNSSIGPNDLSKRKQRKKQIHEYEGVLEEY YSQGTTVVNSISAQIYSLLSAIGETNLSNLWLNILGTTSLDIAYAQVYNRLYPLLQDEVKRLTPSSRNSVKTPDTLTLNI QPDYYLFLLRHSSLYDSFYYSNYVNAKLSLWNENGKKRLHKMFARMGIPLSTAQETWLYMDHSIKRELGIIFDKNLDRYG LQDIIRDGFVRTLGYRGSISASEFVEALTALLEVGNSTDKDSVKINNDNNDDTDGEEEEDNSAQKLTNLRKRWVSNFWLS WDALDDRKVELLNRGIQLAQDLQRAIFNTGVAILEKKLIKHLRIYRLCVLQDGPDLDLYRNPLTLLRLGNWLIECCAESE DKQLLPMVLASIDENTDTYLVAGLTPRYPRGLDTIHTKKPILNNFSMAFQQITAETDAKVRIDNFESSIIEIRREDLSPF LEKLTLSGLL Q08032 DNA polymerase alpha-binding protein Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.104543391 3 Saccharomyces cerevisiae LEVO MVSVIDKLVFDFGGKTLVSLAPDNNTLCVANKNGLTKILKTNNPEEEPETLDSSKLVSSIKCYSNSHFLMTTMQGDALRY NIDSSQEELLARFALPLRDCCVIHSGKMAVFGGDDLELILLELDDETHKKHAIKIDEQVSQISYNSQMNILAVSMINGKV QIFSLTSTIPNKVHELNDYIVANSYDDTHRDKILSNMMDDIDKDNDNDLSETADPDENNVADPEFCAANRICTRVAWHPK GLHFALPCADDTVKIFSIKGYSLQKTLSTNLSSTKAHFIDLQFDPLRGTYIAAVDLNNKLTVWNWETSEIHYTREFKRKI TNIAWKIQADSKTLDLVLGTWSGSIAIVQNLAESVVSNIPDQSVAESSTKHGLFVDSESDLENLEGNDDINKSDKLFSDI TQEANAEDVFTQTHDGPSGLSEKRKYNFEDEEDFIDDDDGAGYISGKKPHNEHSYSRVHKTHSFPISLANTGKFRYMPFS PAGTPFGFTDRRYLTMNEVGYVSTVKNSEQYSITVSFFDVGRFREYHFEDLFGYDLCFLNEKGTLFGQSKTGQIQYRPHD SIHSNWTKIIPLQAGERITSVAATPVRVIVGTSLGYFRSFNQFGVPFAVEKTSPIVALTAQNYRVFSVHYSQFHGLSYSL SELGTSSKRYYKRECPLPMSLPNINSDMKKDANLDYYNFNPMGIKSLFFSSYGDPCIFGSDNTLLLLSKWRSPEESKWLP ILDSNMEIWKMSGGKETTDIHVWPLALAYDTLNCILVKGKHIWPEFPLPLPSEMEIRMPVFVKSKLLEENKAILNKKNEI GADTEAEEGEEDKEIQIPVSMAAEEEYLRSKVLSELLTDTLENDGEMYGNENEVLAALNGAYDKALLRLFASACSDQNVE KALSLAHELKQDRALTAAVKISERAELPSLVKKINNIREARYEQQLK Q01454 DNA fork, leading-strand template synthetic construct 0.026396835999999996

Cy3-label at 5'-end. Five phosphorothioate backbone linkages between residues at the very 3'-end. The dT 16 residues from the 5'-end was biotinylated.

1 (DT)(DA)(DG)(DA)(DG)(DT)(DA)(DG)(DG)(DA)(DA)(DG)(DT)(DG)(DA)(DT)(DG)(DG)(DT)(DA) (DA)(DG)(DT)(DG)(DA)(DT)(DT)(DA)(DG)(DA)(DG)(DA)(DA)(DT)(DT)(DG)(DG)(DA)(DG)(DA) (DG)(DT)(DG)(DT)(DG)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT) DNA DNA fork, lagging-strand template synthetic construct 0.018524886999999997

dT (residue 47) biotinylated.

1 (DG)(DG)(DC)(DA)(DG)(DG)(DC)(DA)(DG)(DG)(DC)(DA)(DG)(DG)(DC)(DA)(DC)(DA)(DC)(DA) (DC)(DT)(DC)(DT)(DC)(DC)(DA)(DA)(DT)(DT)(DC)(DT)(DC)(DT)(DA)(DA)(DT)(DC)(DA)(DC) (DT)(DT)(DA)(DC)(DC)(DA)(DT)(DC)(DA)(DC)(DT)(DT)(DC)(DC)(DT)(DA)(DC)(DT)(DC)(DT) (DA) DNA Topoisomerase 1-associated factor 1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.141296875 1 Saccharomyces cerevisiae LEVO MSADLQQGTTNAADFSLTVLRARIALLATAIGGPDYTSQIDPPPYKLGDDCLACLKDLKRWFKLVDDQQKRWDVAMAVAE YRILTDDLLPILIDWENKCSLAAKLAKNNPDHEEFRNKAYYDKIALNCLQLLVLMTWPLIVTEQSSSNQITLYGELKKHQ LVYKKTILSMESGKVLRAAIRLALDVIKIDRLSRTPRDNMVLKLVLNFFRNVIAIEPGEFTINTKKSMPKKGITSIDTLP PNVSMDDISLNTVISSFHKNKVFGFLLTLTSSLSKEFDQDFINIPLLEIMFYFTKDVNQELLFPRQFETGTHSKVVNKNE SSSANNIVTSAGFELSKLLQKEHQMRKNVIKHTSARHSRFGGLLSIQTPDKTRLTVSGSQALVDEKIALQKLDDSKKWNK RIIKKHQSVAAEGLPNSLLNSQTGKAIFFTESNGKHFKEFINNFIDSGFNILLHSVTNYFTTEQDRMVTLEQVEYLLFFA WFVKYQLLRSKIDNSADIKQVSEALKEVTFILVSSLLRSAYDLKNWTVTHAGMIAFNELLNLVSRTKAAQEEDSTDIEFI VSRLFSDERIQLLSNLPKIGSKYSLQFMKSCIELTHSVLKVLEQYSDDKTLVIEGKSRRQKKFNISEGDITKLIEEENVD RDEALDILTSSLRSIEVNFQKVQANYMTEPVIETYINFLERFRELEDDSIKKVFSFFHRVFVQAKEQALLFRFDLIILLR EMLSPDGLDRMSRSRKYVSQFSDYFLARLKKRLKKSPAWFVGLLFPPLHNSEVGFYQRYGEYNVLNNESMYAAPASQFKP IPDEEALPPSILLDMKYGVLVSTLLDDGKTELLDQLLKHITHTLDIFKSWLTVNVNAGKETVNPPNEYFTLTGVLNNDPI FKDKDYRALLLLIGYSIPRKINEPCFLPGTVEVSDLTVSCELVKKYLSTPFETPNGLPSSSYLLRVRSEKDSFSHNEQDG WEGDDDYDYNDPYIVPDDQILSKSDAAYFKDLDNNASDKLKGTKFSKGIARSKKKDKRKRRKGEAKTNLPMFGDQDDERP QTVRERHGVFSKEFISDSEDDEDLMNPIFFENETYMRWLLDKNNGQLTEDRYIQFAKFAAERMNNGGVVTGDYTSLFGGS IPSIESIRATESSSFAPDKSLISLASHVASEMSIFDVNNNNNNQLSDDDVNSESRNSLGSSQPSNSQNMFQSEVYSRKES TKRSLEASAADESDEDEEAIRLFGKKSRVVLSQGDSDD P53840 Chromosome segregation in meiosis protein 3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 0.03640258999999999 1 Saccharomyces cerevisiae LEVO MDQDFDSLLLGFNDSDSVQKDPTVPNGLDGSVVDPTIADPTAITARKRRPQVKLTAEKLLSDKGLPYVLKNAHKRIRISS KKNSYDNLSNIIQFYQLWAHELFPKAKFKDFMKICQTVGKTDPVLREYRVSLFRDEMGMSFDVGTRETGQDLERQSPMVE EHVTSAEERPIVADSFAQDKRNVNNVDYDNDEDDDIYHLSYRNRRGRVLDERGNNETVLNNVVPPKEDLDALLKTFRVQG PVGLEENEKKLLLGWLDAHRKMEKGSMTEEDVQLIQSLEEWEMNDIEGQHTHYDLLPGGDEFGVDQDELDAMKEMGF Q04659 ZINC ION 6.5409e-05 5 ZN PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER 0.000506196 3 ANP MAGNESIUM ION 2.4305e-05 3 MG singleParticle particle 0.5 7.5 25.0 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 150.0 NaOAc sodium acetate 0.5 TCEP tris(2-carboxyethyl)phosphine 0.1 AMP-PNP adenylyl-imidodiphosphate 0.7 Mg(OAc)2 magnesium acetate 0.005 TWEEN20 Polyoxyethylene (20) sorbitan monolaurate Quantifoil R2/2 COPPER 400 CARBON CONTINUOUS 5.0 GLOW DISCHARGE 5 AIR

15 mA

ETHANE 80 277.15 HOMEMADE PLUNGER

Three microlitres of sample was applied on a grid and incubated for 15-30 s at 4 degC before manually blotting with filter paper for 10 s and plunge-freezing in liquid ethane..

In vitro reconstitution from individual components: CMG, Csm3-Tof1, Mrc1, Ctf4 and a DNA fork

FEI TITAN KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 50.0 2.7 1.4000000000000001 0.5 2.6 3.0 130000.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN GIF Bioquantum 20 GATAN K2 QUANTUM (4k x 4k) COUNTING 1-20 2 6682 7.0 37.0 1 632000 5U8S 1 C1 FOURIER SPACE 3.7 FSC 0.143 CUT-OFF RELION 3.0.6 35000 MAXIMUM LIKELIHOOD RELION 3.0.6 MAXIMUM LIKELIHOOD RELION 3.0.6 RELION 3.0.6 emd_10227.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 360 360 360 0 0 0 360 360 360 377.64 377.64 377.64 90.0 90.0 90.0 X Y Z -0.033392858 0.06839484 0.00003796763 0.00300201 1.049 1.049 1.049 0.018 AUTHOR ::::EMDATABANK.org::::EMD-10227:::: Conformation1 CMG-Csm3-Tof1-Mrc1-Ctf4 and a DNA fork 5U8S PDB experimental model 5MQI PDB experimental model 4C8H PDB experimental model OTHER FSC 0.5 REAL 20.0